Method of searching for gene encoding nuclear transport protein

Abstract

Each gene in a group comprising many genes is cloned into an expression vector so that its gene product should be expressed as a fusion protein with a protein emitting self-fluorescence to construct a gene expression library. The gene expression library is introduced into cells of two groups to express the fusion proteins in the cells, and only one cell group is stimulated by a drug or the like. Then, cells in which the fusion proteins are localized in the nuclei or their nuclei are isolated from cells of the two groups, respectively. By comparing these cells, genes coding for proteins that are transported into the nuclei specifically in response to a certain stimulus when the stimulus is applied to the cells are retrieved.

1. A method for searching for a gene coding for a protein which is transported from cytoplasm into a nucleus through a nuclear pore in a eukaryotic cell, comprising the steps of:
 (a) introducing a library of genes coding for fusion proteins of a marker protein whose localization in a cell can be detected and a part or whole of a search object protein into eukaryotic cells to allow expression of the genes in the transgenic cells so that the fusion proteins are produced, wherein one or more genes coding for a fusion protein autonomously transporting into the nucleus with no stimulus are eliminated from the library beforehand; 
 (b) dividing the transgenic cells into two groups and stimulating only cells of one group, wherein the stimulation transports a nuclear transport protein into nuclei of the cells, thereby transporting the fusion protein comprising a nuclear transport protein into nuclei of the cells; 
 (c) detecting localization of the fusion proteins in the cells by using the marker protein in each of the stimulated cell group and the unstimulated cell group and isolating cells in which the fusion proteins are localized in the nuclei or their nuclei; and 
 (d) recovering genes coding for a fusion protein from the isolated cells or their nuclei, and comparing genes recovered from the stimulated cell group and genes recovered from the unstimulated cell group. 
 
     
     
       2. The method according to  claim 1 , comprising, after the step (d), a step of identifying a gene recovered only from the stimulated cell group or a gene recovered only from the unstimulated cell group. 
     
     
       3. The method according to  claim 1 , wherein, in the step (c), operations of detecting localization of the fusion proteins in a cell based on the marker protein and isolating cells or nuclei in which localization of the fusion proteins is observed in the nuclei are repeated twice or more for isolating cells or nuclei. 
     
     
       4. The method according to  claim 1 , wherein the marker protein is a protein emitting self-fluorescence, a protein catalyzing color development reaction, an antigen peptide that can be detected with an antibody or a peptide that can specifically bind to another substance. 
     
     
       5. The method according to  claim 4 , wherein the protein emitting self-fluorescence is a green fluorescent protein. 
     
     
       6. The method according to  claim 1 , wherein, in the step (b), the transport of the nuclear transport protein into the nuclei is stimulated by means selected from the group consisting of chemical substance, a physiologically active substance, environmental change and contact with other cells. 
     
     
       7. The method according to  claim 1 , wherein the cells or their nuclei are isolated by cell sorting, flow cytometry, microdissection or microscopic observation. 
     
     
       8. The method according to  claim 1 , wherein, in the step (c), permeability of the cell membranes is increased so that the fusion proteins in the cytoplasm should be flown out of the cells prior to the isolation of the cells. 
     
     
       9. The method according to  claim 1 , wherein, in the step (c), the cell membranes of the cells are disrupted and then the nuclei are isolated. 
     
     
       10. The method according to  claim 1 , wherein, in the step (d), the genes recovered from the stimulated cell group and the genes recovered from the unstimulated cell group are compared based on sequence analysis of full length or a part of a region coding for a search object protein for the genes. 
     
     
       11. The method according to  claim 1 , wherein, in the step (a), one or more genes coding for a fusion protein with an extranuclear protein, which is never transported into the nucleus, are eliminated from the gene library beforehand. 
     
     
       12. The method according to  claim 1 , wherein, in the step (a), the search object protein is a protein which regulates gene transcription. 
     
     
       13. The method according to  claim 1 , wherein, in the step (a), the fusion proteins further include a nuclear export signal.